This research program is concerned with the elucidation of the mechanism of DNA replication of the small Bacillus subtilis bacteriophage phi 29. The genome of phi 29 is a linear, non-redundant duplex DNA molecule of about 18,000 base pairs. At present the mechanism of initiation and termination such linear DNA molecules are not understood. Our study of replicative phi 29 DNA will be extended to an investigation of the origin and terminus of DNA replication, by pulse labeling DNA during its synthesis and determining the distribution of radioactivity in restriction endonuclease fragments. We also propose to isolate replicative intermediates by density labeling and analyze them by sedimentation, electrophoresis and electron microscopy. In addition, we propose to develop an in vitro DNA synthesis system from phi 29 infected cells to investigate the molecular mechanism of linear DNA replication.